Suppression of Forkhead Box Protein 01 (FOX01) Transcription Factor May Promote Adrenocortical Tumorigenesis
Authors
Adam Stenman1, 2, 3, Timothy Murtha1, 2, Reju Korah1, 2, Tobias Carling1, 2
Affiliations
1 Yale Endocrine Neoplasia Laboratory, Yale School of Medicine, New Haven, Connecticut, USA
2 Department of Surgery, Yale School of Medicine, New Haven, Connecticut, USA
3 Department of Oncology-Pathology, Karolinska Institu- tet, Stockholm, Sweden
Key words
adrenal cortical adenoma, adrenocortical carcinoma, tumorigenesis, FOXO
received 31.03.2017 accepted 25.04.2017
Bibliography
DOI https://doi.org/10.1055/s-0043-110143 Published online: 2017 Horm Metab Res
@ Georg Thieme Verlag KG Stuttgart . New York ISSN 0018-5043
Correspondence
Tobias Carling, MD, PHD, FACS Section of Endocrine Surgery Department of Surgery Yale Endocrine Neoplasia Laboratory Yale School of Medicine 333 Cedar Street, TMP FMB130A, P. O. Box 208062 New Haven, CT 06520, USA Tel .: + 1/203/737 2036, Fax: + 1/203/737 4067 tobias.carling@yale.edu
Supporting Information for this article is available online at http://www.thieme-connect.de/products.
ABSTRACT
Despite recent comprehensive genetic analyses, molecular evidence for a pathophysiological continuum linking benign adrenocortical adenoma (ACA) and highly aggressive adre- nocortical carcinoma (ACC) is still elusive. Using human tumor samples and the established ACC cell line SW-13, this study investigated potential regulatory roles for FOXO transcription factors, in modulating adrenocortical tumorigenesis. Adre- nocortical tumor specimens (20 ACAs, 10 ACCs, and 9 normal adrenal tissue samples) obtained from 30 patients were ana- lyzed for ubiquitously expressed FOXO transcription factors, FOXO1 and FOXO3 using qRT-PCR and immunohistochemistry. The SW-13 ACC cells were used to study the phenotypic effects of FOXO regulation in vitro. While FOX03 expression remained unchanged in ACCs, FOXO1 expression was found to be signi- ficantly downregulated in 19/20 ACAs and 9/10 ACCs (p<0.0001 and p<0.05, respectively), suggesting a global role for FOXO1 suppression in promoting and maintaining adre- nocortical dedifferentiation. Silencing of FOXO1 in SW-13 cells resulted in significant loss of viability (p<0.001) mediated by apoptosis as determined by quantitative Annexin V immunoflu- orescence analysis (p<0.01). FOXO1 silencing also augmented the migratory behavior of SW-13 cells (p<0.0001), suggesting distinct roles for FOXO1 in promoting viability and controlled motility of adrenocortical cells.
Introduction
Adrenocortical tumors (ACTs) are very common with a population prevalence between 1.4% and 8.7% according to autopsy studies [1,2], and the majority of ACTs are benign adrenocortical adeno- mas (ACA) [3]. Adrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine malignancy with an incidence of 1-2 affect- ed patients per million per year [4], and is responsible for approxi- mately 0.2 % of all cancer deaths in the United States [5]. The ther- apeutic options are presently limited, and despite complete surgi- cal removal, the majority of patients ultimately succumb to their disease. ACCs occur more often in females [6], while ACAs have a
near-equal gender ratio [2]. ACTs can present with clinical signs of hormonal hypersecretion due to excess aldosterone, cortisol, or androgens, or can also be hormonally silent [7].
The genetic underpinnings of ACTs are heterogeneous and vary depending on tumor characteristics; benign, malignant, or hor- mone secretion status. The alterations in ACA have been elucidat- ed to a greater extent than ACC, and have been linked to variations in the cAMP signaling pathway [8], including mutations in the PRK- ACA gene encoding protein kinase A (PKA) [9, 10], and in genes regulating voltage-gated potassium channels [11]. In contrast, ACC has been associated with functional alterations in TP53, TGF-B,
| Variable | ACC | ACA |
|---|---|---|
| Total (n) | 10 | 20 |
| Age + SD | 55.5± 10.35 | 47.6± 13.2 |
| Male | 2 | 6 |
| Female | 8 | 14 |
| Stageª | ||
| I | – | n/a |
| II | 2 | n/a |
| III | 5 | n/a |
| IV | 3 | n/a |
| Tumor diameter, cm, mean (range) | 11.3 (6.0-14.0) | 3.3 (1.1-6.5) |
| Hormonal status | ||
| Non-hyperfunctional | 3 | 7 |
| Cortisol | 4 | 6b |
| Aldosterone | 0 | 6 |
| Estradiol | 1 | 0 |
| Multiple | 1 | 1 |
| Unknown | 1 | 0 |
a Staging criteria per American Joint Committee on Cancer (AJCC), 7th edition. b Three of 6 patients with subclinical Cushing’s
syndrome. SD: Standard deviation; n/a: Not available
Wnt, and IGF2 [12, 13]. Most ACCs are sporadic, but many are dis- covered in the context of rare tumor syndromes, that is, Li-Frau- meni syndrome, caused by a mutation in the TP53 gene [14], mul- tiple endocrine neoplasia type 1 (MEN1), caused by a mutation in the MENIN gene, and Beckwith-Wiedemann syndrome with a com- plex genetic pathogenesis [3]. Despite recent discoveries, mostly through comprehensive genetic analyses [15], the molecular eti- ology of sporadic ACCs remains incompletely understood [16]. Moreover, despite the common tissue origin, no pathological con- tinuum has been proven through benign ACA to malignant ACC.
Both Wnt and the transforming growth factor beta (TGF-B) path- ways are known to have roles in the development and function of the adrenal cortex and are frequently found abrogated in ACTs [13]. Moreover, inhibin and activin, members of the TGF-B family, are implicated as differentiation-promoting factors that support de- velopment of the adrenal cortex [17]. The forkhead box transcrip- tion factor class O (FOXO) include FOXO1, FOXO3, FOXO4, and FOXO6. These transcription factors are regulated by multiple growth factors, including insulin-like growth factor (IGF), through phosphorylation by AKT/PKB [18, 19]. In addition, deregulated cross-talk between TGF-B and FOXO signaling has been shown in multiple cancers [20-23]. Since aberrant TGF-ß signaling has been shown to promote ACT tumorigenesis, this study sought to inves- tigate a potential modulatory role for FOXO1, a known TGF-ß sig- nal transducer, in blocking adrenocortical dedifferentiation and/or promoting malignancy.
Materials and Methods
Tissues, cell, and nucleic acid preparations
Following approval by the Yale University Institutional Review Board and obtaining written informed consent from, 30 ACT samples (20 ACAs and 10 ACCs), together with 9 normal adrenal specimens ad- jacent to ACAs, were collected from 30 patients (8 males, 22 fe- males). Patient demographics and associated clinical and patho- logical findings are detailed in » Table 1. All adrenal tissues were histologically confirmed by an experienced endocrine pathologist prior to immunohistochemistry (IHC) and extraction of RNA. The established ACC cell line SW-13 (human, female cell line, from and authenticated by ATCC), was used for in vitro phenotype analyses. Total RNA was extracted from frozen adrenal samples and SW-13 cells using the RNeasy Plus Mini kit (Qiagen), and the quantity and qualities of the preparations were determined using a NanoDrop spectrophotometer (ND-1000; Thermo Fisher Scientific). cDNA was synthesized from 400 ng of total RNA using the iScript com- plementary DNA synthesis kit (Bio-Rad). Immunohistochemical analyses were performed using representative formalin-fixed, par- affin-embedded tissue from primary tumors cut 5 M thick, ob- tained from the Yale Pathology Tissue Services.
FOXO gene expressional analysis
Gene expression levels of the ubiquitous FOXO transcription fac- tors FOXO1 and FOXO3 were estimated in an exploratory cohort of ACCs and normal adrenal samples (n = 12) followed by a confirma- tion cohort of ACTs and normal adrenal samples for FOXO1 (total n = 39) using real-time quantitative polymerase chain reaction (qRT-PCR). The qRT-PCR reactions were performed on a CFX96 Re- al-Time System (Bio-Rad) in duplicates using TaqMan PCR master mix with commercially available primer and probe pairs specific to FOXO1 (FAM dyed, assay ID: Hs01054576_m1) and FOXO3 (FAM dyed, assay ID: Hs00818121_m1). The housekeeping gene RPLP0 (FAM dyed, assay ID: Hs99999902_m1) served as an endogenous control. The gene expression data shown in this study represents one of 2 independent experiments with similar results for all cases. Relative gene expression of all tissue samples and SW-13 experi- ments were calculated using the Livak method [24]. Gene expres- sion levels for individual ACT samples were relatively quantified to the mean expression of all normal adrenal samples. Gene expres- sion levels for si-RNA treated SW-13 samples were compared to the expression of the parental SW-13 cells. Downregulation was con- sidered as relative expression < 1, after normalization of all normal adrenal samples to a value of 1.0.
Immunohistochemistry (IHC)
A total of 9 representative ACTs (4 ACCs and 5 ACAs) were analyz- ed using a standard IHC protocol [25], targeting a FOXO1 epitope with an anti-FOXO1 (ab179450; Rabbit monoclonal from Abcam, dilution 1:100) antibody (incubated overnight at 4 ℃) followed by HRP-linked anti-Rabbit secondary antibody incubation (Santa Cruz Biotechnology, Inc., dilution 1:200) and 3,3’-diaminobenzidine (DAB) staining (Thermo Fisher Scientific). Sections were counter- stained in hematoxylin (VWR Scientific) and mounted using immu- nohistomount (Santa Cruz Biotechnology) prior to photomicrog- raphy at 400 x . Adjacent histologically normal adrenal cortex sam-
ples served as positive controls and omission of primary antibody as negative control.
Cell culture, siRNA transfection, and western immunoblot detection
The established human ACC cell line SW-13 was grown under ster- ile conditions in DMEM as previously described [25]. For siRNA transfection, 0.2 × 106 cells/well were seeded in 6-well plates one day prior to transfection in order to reach ~80% confluence. Cells were transfected with mock medium (Lipofectamine-2000, Ther- mo Fisher Scientific), negative scrambled siRNA (Origene; 20 nM), or FOXO1 (Human) - 3 unique 27mer siRNA duplexes targeting FOXO1 (Origene; 20 nM) in Opti-MEM (Thermo Fisher Scientific), following the protocol from the manufacturer of lipo- fectamine-2000 (Thermo Fisher Scientific). Transfection medium was replaced with complete medium after 6 h of transfection and cells were lysed after 24 or 48 h for RNA or protein extraction, re- spectively. Three different siRNAs (siA, siB, and siC; Origene) tar- geting 3 distinct sites on FOXO1 mRNA were tested. SiA was select- ed for phenotype studies based on the marked FOXO1 downregu- lation as determined by qRT-PCR and confirmed by Western immunoblot (Fig. 1S). The Western Blot method, as described pre- viously [26], was performed using the anti-FOXO1 (ab 179450; Rab- bit monoclonal, Abcam) antibody and GelCode Blue Safe Protein Stain (Thermo Fisher Scientific) served as the loading control. Trans- fection validation and silencing efficacy are shown in the Supple- mentary Material, Fig. 1S.
Cell viability, motility, and apoptosis detection assay
The viability and motility assay data shown in this study denotes triplicate samples assayed from one of 2 independent experiments. Total cell number and viability were calculated with the Trypan Blue exclusion assay (Thermo Fisher Scientific) and manual counting by 2 researchers independently, using a hemocytometer (Hausser Sci- entific).
The migratory potential of the cells was measured using modi- fied Boyden Chambers (BD Biosciences); 1 x 105 cells were allowed to migrate through 8 um pores from the upper chambers contain- ing serum-free medium to the lower chambers containing com- plete medium (DMEM containing 10% FBS). After 4h, cells that had migrated to the lower side of the membranes were fixed in 3.7 % formaldehyde for 10 min, stained with 0.05 % crystal violet for 30 min and counted using a light microscope at 100 x.
The rate of apoptosis was determined by quantitative Annexin V immunofluorescence analysis using the ApoDETECT, Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). Cells were stained simultaneously with Annexin V-FITC (detecting phos- phatidylserine in an early stage of apoptosis) and propidium iodide (PI) to identify and quantify apoptotic cells using fluorescence mi- croscopy.
Statistical analysis
As the gene expression of ACT samples had a non-normal distribu- tion, a 2-tailed Mann-Whitney U test (MWU) was applied to inves- tigate potential correlations between FOXO1 gene expression in ACC, ACA, and normal adrenal samples. A 2-tailed Student’s t-test (TT) was used when comparing the cell count means for the indi-
vidual treated/nontreated SW-13 cells. p-Values <0.05 were con- sidered to be significant in all cases.
Results
FOXO1 expression is downregulated both in ACAs and ACCs
To test potential roles for FOXO transcription factors in mediating global signaling dysregulation observed in ACTs [3, 13], first we de- termined the expression levels of ubiquitous FOXOs; FOXO1 and FOXO3 [27]. While FOXO3 expression remained stable between normal and ACCs ( Fig. 1a), similar to the expression pattern of the house-keeping gene RPLP0, FOXO1 expression appeared to be downregulated. Using qRT-PCR and IHC for detection of mRNA and protein expression respectively, we tested whether the expression of FOXO1 was reduced significantly in benign and malignant ACTs compared to normal adrenal samples. Of 20 ACAs, 19 were found to have reduced mRNA expression of FOXO1 (mean 0.36±0.23) with one ACA sample (# 9) showing relative FOXO1 mRNA expression of 1.04. This correlation was found to be statistically significant com- pared to normal adrenal samples (p<0.0001, MWU) ( Fig. 1b). Regarding the ACCs, 9/10 had reduced expression of FOXO1 (mean 0.63 ± 0.49) with one sample (# 25) showing relative FOXO1 ex- pression of 1.84. Similar to benign ACA’s, FOXO1 expression silenc- ing in malignant ACCs was also found to be statistically significant (P=0.026, MWU) ( Fig. 1b). However, there was no statistical dif- ference between ACAs and ACCs (p=0.1, MWU; > Fig. 1). The FOXO1 protein expression analysis correlated with reduced FOXO1 mRNA expression levels as determined by IHC in 9 randomly select- ed ACA and ACC samples, with negative FOXO1 nuclear staining in the ACTs and positive nuclear immunostaining in all normal adre- nal cortical samples ( Fig. 1c).
FOXO1 silencing mediates significant loss of cell viability via apoptosis
In order to test whether FOXO1 plays a distinct role in ACT tumor- igenesis, we studied the effects of focused FOXO1 silencing in in vitro in the established human ACC cell line SW-13. While main- taining an intact Wnt signaling pathway, the SW-13 cells harbor a dysregulated TGF-B signaling [28] and hence is ideally suited to test the functional role of FOXO1, a known downstream target of TGF-฿ signaling [21]. A previously established transient siRNA silencing method [29], was used to test whether suppressing endogenous FOXO1 in isolation would affect the malignant properties of the ACC cells. The SW-13 cells were treated with 3 different siRNAs and treatment with 2 of 3 siRNAs resulted in reduced FOXO1 expres- sion with >60% efficiency (siA and siC; Fig. 1S). SiA was selected for further phenotypic analysis. The effect of FOXO1 suppression on cell viability was measured after treating cells for 24h with siRNA (siA) or negative scrambled siRNA (SW-NS). Starting with 0.2 x 106 cells/well for all treatments, the total number of cells/well after 24h of treatment were 0.67 x 106 for the mock-treated (Lipofectamine only) cells (SW-L), 0.5 x 106 for the negative scrambled siRNA treat- ed cells (SW-NS) and 0.29 x 106 for the FOXO1 silenced cells (SW- siA; p<0.001, TT for both SW-siA vs. SW-NS and SW-siA vs. SW-L; Fig. 2a). Hence, the loss of viability consequent to FOXO1 silenc- ing was found to be statistically significant.
a
b
2.5
2.0
P < 0.05
1
*
P < 0.0001
FOXO3 relative expression
2.0
FOX01 relative expression
1.5
1.5
1.0
1.0
0.5
0.5
0
0
Normal
ACC
Normal
ACA
ACC
c
Normal adrenal cortex
ACA
ACC
~10um
To test whether the loss of viability is due to programmed cell death, an Annexin V-FITC Apoptosis Detection Kit and immunoflu- orescence microscopy were used. The proportion of apoptotic cells was 2.8 % for SW-L (Lipofectamine only), 3.1 % for the SW-NS (neg- ative scrambled siRNA treated cells), and 9.9% for the SW-siA (FOXO1) treated cells (p<0.01, TT for SW-siA vs. SW-NS). Hence, the cell death observed consequent to FOXO1 suppression can be primarily attributable to apoptosis ( Fig. 2b, 3), suggesting a role for FOXO1 in promoting SW-13 ACC cell survival.
Silencing of FOXO1 results in increased migratory behavior of ACC cells
SW-13 cells possess robust motile behavior reflecting their malig- nant phenotype [26]. To test whether FOXO1 plays a role in ACC cell’s aggressive migratory behavior, we compared the motility of
SW-13 cells subsequent to siRNA-mediated FOXO1 silencing. The average number of migrated cells was compared in SW-L, SW-NS, and SW-siA treated SW-13 cells. FOXO1-suppressed SW-13 cells migrated to a greater extent with a 3.5-fold increase in the number of migrated cells after 4 h compared to the control treatment sam- ples. Significant correlations were found for both SW-siA vs. SW-NS and SW-siA vs. SW-L (p<0.0001, TT; > Fig. 2c), suggesting a role for FOXO1 in modulating the adhesion/spreading or motility kinet- ics of SW-13 cells.
Discussion
The diversity of genetic, epigenetic, and signaling disarray found in ACTs makes the search for a precise molecular etiology of adreno- cortical tumorigenesis very challenging. Despite recent rigorous and comprehensive genetic analyses, it is still unclear whether a benign
a
b
c
800000
12
P <. 01
2500
P <. 0001
700000
P <. 001
10
2000
600000
8
500000
Cell # per ml
% of apoptotic cells
Average # of migrated cells
1500
400000
6
300000
1000
4
200000
T
500
L
2
I
1
100000
0
SW-L
SW-NS
SW-siA
0
SW-L
SW-NS
SW-siA
0
SW-L
SW-NS
SW-siA
Downloaded by: Cornell. Copyrighted material.
cortical lesion, such as an ACA, harbors malignant potential and is able to transform into an ACC. The near-complete disconnect be- tween the known genetic events in ACAs and ACCs may in fact sug- gest that they could be independent entities. Current knowledge re- garding ACT development has implicated alterations in specific ge- netic drivers abrogating ion channel functions in ACAs, while ACCs present a picture of genetic and signaling chaos. It is not known if the initial genetic aberrations in ACA are followed by secondary events, and perhaps the accumulation of multiple subtle changes lead to malignant transformation. We hypothesized that the discrep- ancies in transcriptional regulation observed in ACCs may constitute one of the subtle but potentially dominant changes shared by ACTs of diverse origin. The FOXO transcription factors belong to the fam- ily of one of such factors that has been shown to be shared by mul- tiple signaling pathways as well as found to be dysregulated in mul- tiple cancers [20-23]. Particularly interesting are potential roles of FOXO1 and FOXO3, 2 ubiquitously expressed FOXO family members [30]. Moreover, dysregulation of TGF-ß signaling has been shown to modify a variety of cell behaviors including cell proliferation, migra- tion and apoptosis through modulating the transcriptional activity of FOXO transcription factors including FOXO1 [17, 19,31]. There- fore, we chose to investigate potential dysregulation of FOXO1 and FOXO3 in the context of ACTs.
Our finding that expression of FOXO1 is specifically and signifi- cantly downregulated in 28/30 ACTs compared to the normal ad- renal cortical tissue strongly suggests a global role for FOXO1 in promoting differentiation and/or tumor suppression downstream to multiple overlapping signal transduction pathways in the adre- nal cortex. Whether the robust FOXO1 expression in normal adre- nal cortex observed in this study and by others [32], implies specif- ic roles in structural/functional differentiation or adrenocortical endocrine homeostasis, needs to be studied further. It is also inter- esting to note that FOXO3 remained unchanged in ACCs despite significant silencing of FOXO1, potentially suggesting non-over- lapping roles for FOXO1 and FOXO3 in adrenal physiology. Despite the relatively small sample size, the results in this study specifical- ly suggest a role for early and persistent FOXO1 suppression in the origin, maintenance, and/or progression of both ACA and ACC. However, it still remains to be seen whether the observed down- regulation represents an earlier event promoting dedifferentiation or a later event promoting migration and malignancy. The down- regulation of FOXO1 in both ACA and ACC argues against the prev- alent assumption that ACAs and ACCs are independent entities, and instead provides support that there is a continuum of signal- ing transformations shared by both tumor types - potential evi- dence of a molecular lineage that has not been previously described in ACTs.
SW13 - Negative Scrambled (SW-NS)
SW13-FOX01 siRNA (SW-siA)
In vitro RNAi-promoted silencing of FOXO1 in SW-13 cells fur- ther support FOXO1’s diverse roles in adrenocortical carcinogen- esis that are probably signaling-context dependent. SW-13 cells harbor overlapping and potentially interfering dysregulated TGF- ß, aberrant TP53, and differentiation-promoting Wnt signaling cas- cades. While it is conceivable, that aberrant TGF-ß signaling or de- regulation of the TP53 network may necessitate cell-survival by AKT via FOXO1 in SW-13 cells [19], this scenario is not consistent with the near-universal suppression of FOXO1 in ACTs. It should also be noted that TP53 mutations represent only a minor portion of sporadic ACT’s and hence SW-13 signaling may not accurately reflect the majority of ACTs. The observed augmentation of migra- tory behavior might appear to contradict the tumor suppressor role implied by loss of viability consequent to FOXO1 downregulation. Superficially, the conflicting behavioral outcomes of FOXO1 silenc- ing in SW-13 cells may appear to reflect a purely cell-specific in vitro phenomenon. On the other hand, the increased migratory behav- ior could potentially represent a partial relief of motility restraint imposed by Wnt signaling executed via FOXO1. The diverse effects on in vitro behavior of ACC cells also may attest to the expected promiscuous affinity of transcription factors in general, regulated by genetic and epigenetic upstream events. Further experiments targeting isolated downstream signaling events are needed to clar- ify the specific roles of FOXO1 silencing in the promotion and/or progression of ACTs.
In summary, this study highlights a potential role for FOXO1 sup- pression in the molecular etiology and development of ACTs and, more intriguingly, a novel disease continuum between ACA and ACC.
Conflicts of interest
The authors declare that they have no conflict of interest.
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